Blood Investigations: Dr. Maulika Patel

BLOOD INVESTIGATIONS
BLOOD
INVESTIGATIONS
DR. MAULIKA PATEL

 COLLECTION:
Blood is Collected for many different purpose, like
1. Haematological Examination
2. Serological Examination
3. Bio Chemical Examination
4. Culture Examination
5. For Blood Transfusion.
Collection of Blood Specimen
Method of blood collection depends on type of investigation to
be done and the quantity of blood required.
1. Capillary Blood Specimen
Capillary Blood is Obtain when only few drops of blood are
required for investigation. eg.
- Haemoglobin Estimation
- Bleeding and Clotting Time
- Blood grouping and counting
- Preparation of Blood Smear
-> Capillary Blood is collected from
- Tip of the Finger
- Lobe of ear
- Hill or toe in infants
-> Methods
- Equipments: Spirit or 70% Alcohol, Cotton, Needle or Mechanical

gun, Appropriate Pipette, Glass Slide, ETC
DR. MAULIKA PATEL Page 2

->Technique
- The site is cleaned with 70% alcohol or spirit and allowed to dry
- Puncture the site with quick stabbing motion by sterile needle or

mechanical gun.
- The cut should be deep enough so that the blood flows freely
without squeezing the site.
- The first drop should be wiped off with dry gauze and subsequent
drop of free flowing blood are used for sample
- After collection ask the patient to hold the cotton swab in its place
between finger and thumb till bleeding stops.
2. Venous Blood Specimen
Venous blood is collected when investigations required large amount

of blood. Eg
- ESR
- PCV
- Bio chemical, Serological and culture Examination
- Some of Investigation which can be done with Capillary
blood can also be carried out on Venous Sample.
-> Venous Blood is collected from
- Anterior Cubital vein
- Veins on dorsum of
- External jugular or femoral vein in children
- Anterior Fontanelle in infants.

-> Methods:
Equipments: Spirit or 70% alcohol, Cotton, Sterile Syringe & Needle,

Appropriate Containers, Tourniquet Weight.
->Procedure:
- Prepare the patient and equipments
- Apply the Tourniquet ant Select the vein to be
puncture.
- Clean the site with spirit or 70% alcohol and Dry.
- Insert the Needle through the skin and into the vein
- Aspirate blood into the syringe and remove the
tourniquet.
- Place dry cotton swab over the puncture site and
withdraw the needle
- Bring the patient forearm back again his upper arm
while he maintains finger pressure on the cotton at the
puncture site with the other hand.
- Next step is to first remove the needle and than
transfer the blood to suitable container.
a. Plain Test tube is used when investigations to be

done on serum
Eg. Serum Cholesterol, Serum Alkaline Phosphates,
or Immunological test e.g. Widal test, V.D.R.L. test
etc.
b. When examination is to be done on whole blood it is
collected in a container containing suitable
anticoagulant with gentle rotatary motion to allow
thorough add mixture of blood with anticoagulant.
-

ROUTINE INVESTIGATIONS
Total RBC (Erythrocyte) Count

- It gives the number of erythrocytes per cubic millimetre in
circulating blood and also gives an indirect estimate of haemoglobin
in blood. it is determined by haemocytometer.
- The Mature Human erythrocyte is a circular, biconcave,

non- nucleated disc.
- 6.7 to 7.7 mm in diameter.
- Normal value: male – 4.5 – 5.9 million\mm3
female- 4.5-5.1 million\mm3
->Procedure:
- Office and chair side-the test is performed manually
under a microscope by direct counting of the number
of cells in diluted sample of blood confined in a
calibrated chamber of special glass microscope slide
(hemocytometer technique)
- Automated procedure
- The electronic counting of erythrocyte is usually carried
out with equipment such as coulter counter model.
->Interpretation:
- RBCs is reduces in anaemia.

- RBCs increases in polycythaemia.
Erythrocytes Indices
- In evaluation of nature of anaemia, assistance is
obtained by calculating standard indices relating to the
size of RBCs.
Types:
-MCV (mean corpuscular volume)
Average Red cell volume referred to as MCV is expressed in

cubic micron per cell and measured directly or calculated as:
MCV = Hematocrit x 10
RBC in million\mm3
Norma value: 80-96 cu microns
-MCH (mean corpuscular haemoglobin)
The haemoglobin contain of erythrocyte is referred to as MCH,

expressed in pictograms of haemoglobin per cell and is calculated as:
MCH = Haemoglobin Concentration (g\dl) X 10
RBC in million\mm3
Normal value: 27-31 pg
-MCHC (mean corpuscular haemoglobin concentration)
The concentration of haemoglobin is referred as MCHC:
MCHC = Haemoglobin Concentration X 100
Hematocrit

Normal value: 32-36 g\dl
Interpretation:
Types of anaemia MCV MCH MCHC
Microcytic hypochromic Decreased Decreased Decreased
Macrocytic normochromic Increased Increased Normal
Normocytic normochromis Normal Normal Normal
1. Hematocrit (Packed cell volume)

Hematocrit is a measure of volume percent of packed red blood
cells in whole blood.
Normal value: at birth - 54
Adult male - 42- 50 %
Adult female – 36 – 45%
Equipment:
- Two heparinised micro-hematocrit capillary tubes.
- A box of matches and micro-hematocrit centrifuge equipped with

reader and magnifier.
Procedure:
- Blood is obtained by a finger prick or venipuncture and is allowed to

flow by capillary action into two micro-hematocrit tubes ,until each is
about ¾th filled.
- The volume of blood should be free from air bubbles.
- The end of each tube away from the column of blood is sealed by
melting the glass in the flame of a match or small gas jet without
heating the blood.

- The tubes are cooled and placed opposite each other in the slots of
the head of the centrifuge, with sealed ends pointing outwards.
- The centrifuge head covers are closed and the centrifuge timer is
set for 5 minutes.
- The centrifuge is started and it accelerates to 12,500 rpm and stops

automatically at the required time.
- The centrifuge head covers are removed and with the aid of
magnifying lens for greater accuracy, the hematocrit is read by
means of scale incorporated in the head of the instrument.
Interpretation:
- Increased in the value indicates primary and secondary

polycythemia and dehydration.
- Decreased in anaemia.
4. Haemoglobin
-Haemoglobin is the red pigment of blood.
-It is a chromaprotein consisting of two parts. Globin (96%) is a

specific simple protein & Heam (4%) is metalloporphyrin where the
metal is iron (0.34%).
-In human probably two varieties of Haemoglobin:
a. Foetal Haemoglobin (HbF) &
b. Adult Haemoglobin (HbA)
-Normal Value: male- 14 - 17.4 gm\dl
Female- 12.3 to 15.3 gm\dl

Procedure:
-Different methods used are Drabkin’s technique in which

haemoglobin is converted into the stable pigment
cyanmethemoglobin. Drakin’s solution contains potassium cyanide
and ferricynaide and sodium bicarbonate.
-The haemoglobin concentration is usually measured on anti-

coagulated venous blood by reacting the sample with a reagent that
converts the haemoglobin to stable colored products. The
concentration of this colored compound is measured in a
photoelecteic colorimeter in comparison with the standard.
-Other techniques used are sahli’s method (0.1 N hydrogen chloride),

oxyhemoglobin method (0.1 sodium carbonate).
5.WBC (White blood corpuscles):
a. Total WBC count:
Method:
-Total WBC count can be calculated manually with a hemocytometer

or with an automated cell counter.
-To count WBCs in the presence of RBCs, RBCs are lysed by diluting
the blood sample with dilute acetic acid or equivalent reagent
supplied by the manufacture of the automated equipment leaving
the WBCs infact.
Normal value: 4000 – 11000/mm3
Interpretation of total WBC count:
-Leucopenia is an abnormal reduction in the number of WBCs in

peripheral blood circulation.
-Leucocytosis is an abnormal increases in the number of WBCs in

peripheral blood circulation.
Increased in (leukocytosis) Decreased in (leucopenia)
-Infection – acute or chronic -Aplastic anemia
-Leukemia -Influenza, measles and
Respiratory infection
-Polycythemia -Catarrhal jaundice
-Trauma -Early leukaemia
-Exercise, fear and pain -Depression of bone
marrow due to certain drugs
-After general anesthesia -Drug and chemical toxicity,
Shock
b. Differential WBC count:

-The differential leucocyte count is performed by microscopic
examination of a peripheral blood smear or by automated
method.
-There are two main morphologic types of leucocytes in the
peripheral blood.
Types of cell Adult Count
Granulocytes
Neutrophils 50 – 77% 2500 – 7000/
Eosinophil 0 – 4% 50 – 500/
Basophil 0 – 2% 0 – 100/
Agranulocytes
Lymphocytes 20 – 47% 1000 – 4000/
Monocytes 0 – 9% 200 – 800/
Interpretation of differential WBC count:

Increased in Decreased in
1.Neutrophils
-Pregnancy, exercise -Aplastic anaemia
-Infection, inflammation -Cyclic neutropenia
-Neoplasm, Severe hemorrhage -Malignant neutropenia
-Hemolysis and erythroblastosis -Drug induced
Fetails myelosuppression
-Intoxication by drugs and poisons -Early leukaemia
like corticosteroids
2.Eosinophils
-Parasitic Infection -Immune defect
-Allergic disease -Acute stress
-Drug reaction dermatoses -Injection of
like pemphigus, pemphigoid, adrenocorticotrophic
atrophic dermatitis hormone
-Scarlet fever -Typhoid fever
-Aplastic anemia
3.Basophill
-Chronic myelogenous leukaemia -Polycythemia
-Myelofibrosis

4.Lymphocytes
-Lymphocytic leukaemia -Aplastic anemia
-Mumps-German measles -Myelogenous anemia
-Whooping cough
-Chronic infection
-Convalescence from acute infection
5.Monocytes
-Hodgkin disease -Aplastic anemia
-Monocytic anemia -Acute leukaemia
-Malaria- Kala –azar
-Sub-acute bacterial endoccarditis
-Tuberculosis
6.PLATELETS:
Platelets are small 2m to 5m particles released from the cytoplasm of

large multinucleated cells (megakaryocytes) located in the red bone
marrow.
Procedure:
-Counts are performed manually using a special hemocytometer

counting chamber and a phase contrast microscope or by means of
an automated cell counter such as the Technicon autoanalyzer.
Normal value:
Platelet count : 1,50,000 – 4,50,000 /mm3

Interpretation of platelets:
Increased in (Thrombocytosis) Decreased in (thrombocytopenia)
-Chronic myelocytic leukemia -Thrombocytopeniac purpura
-Polycythemia vera -Immune reaction
-Hemolytic anemia -Transfusion
-Trauma -Aplastic anemia
7.BLEEDING TIME:
-It measures the time required for hemostatic plug to form. Lack of
any clotting factor and platelet abnormalities may increases the
bleeding time.
Method:
a. Ivy’s method:-
Equipment: Sphygmomanometer and cuff, 70% alcohol or
spirit, blood lancet, filter paper, stop watch.
Procedure:
-The patient is seated comfortably with his arm supported on
the chair arm r his own thigh.
-The Sphygmomanometer cuff is applied to the patients upper
arm and inflated to 40 mm pressure.
-An area on the palmar (inner) surface of the forearm about
halfway between elbow and wrist that is free of superficial
veins, is located and prepared by scrubbing with an alcohol
soaked swab.

-After the area is air dried, the skin of the prepared area is
tensed and punctured with a blood lancet cutting deep enough
such that the hit is firmly pressed against the skin of the arm.
-The lancet is immediately removed and the time noted on
watch equipped with a seconds hand. Every 30 seconds by the
watch, an edge of filter paper is touched against the drop of
blood that wells up.
-The length of time until the bleeding ceases is the bleeding
time is minutes.
-The area is finally cleaned with a swab slightly moistened in 70
percent alcohol.
Normal Value: 5 to 6 minutes
b. Duke’s method:
-The lobule of ear is punctured & the time is noted. The blood
oozing out is mopped with a filter paper every 30 seconds until
bleeding stops.
-Time form puncturing of ear lobule to stoppage of bleeding is
considered bleeding time.
-Normal bleeding time by Duke’s method is 1 to 5 minutes.
Interpretation: Prolonged time may be due to
-Thrombocytopenia
-Thrombasthenia
7.CAPILLARY FRAGILITY TEST – TORNIQUET TEST AND RUMPEL LEEDE
TEST
Method:
Equipment: Sphygmomanometer and cuff, microscope slide.
Procedure:

-The Sphygmomanometer is applied customarily and the patient’S

systolic and diastolic arterial blood pressure is recorded.
-The pressure in the cuff is reduced to zero and the cuff is reinflated
to a point halfway between systolic and diastolic pressure. This
pressure is maintained for 5 minutes during which the forearm and
hands are examined for development of new petechiae.
-The patient should not be moved vigorously while the cuff is in place
because movement will increase anaerobic muscular glycolysis,
lactate accumulation and thus result in pain.
-After five minutes, the cuff is removed and the patient is allowed to
exercise his arm to restore the circulation in the arm.
Interpretation:
- If any unequivocal petechiae develop distal to the cuff (towards the

hand) after cuff has been applied, the test is recorded as positive.
-It is used to screen for bleeding abnormalities especially where

there is suspicion of deficiency of platelets (thrombocytopenia),
abnormal platelet function (thrombasthenia), a deficiency of
prothrombin (with dicumarol therapy) or damaged, poorly supported
capillary walls (in purpura, scurvy and some collagen disorders).
8. CLOTTING TIME:
It is the time required by blood for coagulation.
Method:
a. Capillary glass tube method:

Sterilize the finger, prick the finger & make the blood to flow
into capillary glass tube about 15cm (6 inches) long after
whipping off first drop Break a small bit of glass tube every ½

minute (30 seconds) until a fine fibrin thread of clotted blood

connecting the broken parts appears while breaking the tube.
Take the period & formation of thread as coagulation time>
-Normal clotting time by capillary method is 3 to 5 minutes.
b. Lee-White method:
1 ml of blood is drown from a vein by dry syringe & placed in
two clean test-tubes, 8 mm in diameter. The tubes are closed
by rubber cork & placed in water bath at 37 C. After 5 minutes
after withdrawn of blood, the first tube is gently tilted 45 at
every 1 minute interval until it can inverted 180 without blood
flowing. This time is recorded & same procedure is repeated
with the second tube. The time for second tube is taken as the
true coagulation time. It is tilted less than the first tube.
-Normal clotting time by Lee-White method is 5 to 10 minutes.
8.CLOT RETRACTION & LYSIS TIME:

Method:
-After determination of clotting till by Lee-White method,
continue to incubate the tubes in waterbath at 37C & Examine
after 1 hour, 24 hours until 72 hours.
-Normally at 37C clot retraction starts within 30 seconds after
blood collection & it is clearly observed at the end of 1 hour.
-The fibrin clot lysis due to fibrinolysis observed at about 72
hours.
Significance:
-In fibrinolytic disease the fibrinolysis is increased.
9.PROTHROMBIN TIME:
Method:
Quick’s method:

-Blood removed from patient, is immediately oxalated so that

none of the prothrombin can change into thrombin. Later, a
large excess of calcium ion & tissue thromboplatin is suddenly
mixed with the exalated blood. Non the time required for
coagulation is known as prothrombin time.
-The prothrombin time gives an indication of the total quantity
of prothrombin in the blood.
-Normal prothrombin time: 12 to 15 seconds.
10.PARTIAL THROMBOPLASTIN TIME:

-It is the time, in seconds, that is required for a clot to form in a
sample of oxalated plasma, to which a partial thromboplastin
reagent and calcium chloride is added.
-Partial thromboplastin which is free of factors V, VII, IX, X, XI,
XII, contains mainly the platelet factor III responsible for
producing plasma thrombopastin. This test, thus reproduces
‘intrinsic’ coagulation mechanism and is a very sensitive
measure of plasma factor deficiencies.
Significance: causes of prolonged PTT are:
-Disseminated intravascular coagulation
-Liver disease
-Administration of heparin or contamination with heparin.
-A circulating anticoagulant.
11. THROMBIN TIME:

Method:
-Add 0.2 ml of citrated plasma in a test tube & place at 37C for
1 minute. Add 0.1 ml of thrombin & calcium chloride mixture &
start the stopwatch. Observe the clot formation & note the
time.
-Normal Thrombin time is 15 to 20 seconds.
Significance: causes of prolonged thrombin time:

-Hypofibrinogenaemia
-Disseminated intravascular coagulation.
-Liver disease
-Presence of heparin.
12.ERYTHROCYTE SEDIMENTATION RATE:

-It is a measure of the rate at which RBCs sediment in a tube of
plasma.
-Method:
a. Wintrobe’s method
b. Wstergren’s method
-Normal value: Male: 0 -15 mm/hr
Female: 0 – 20 mm/hr
-The test is helpful in following the progress of some chronic

infection (tuberculosis and osteomyelitis) as well as disease
characterized by altered globulins such as the collagen disease,
nephritis, rheumatic fever and dysproteinemias.
-The Sedimentation rate may be increased in women with

intra-uterine contraceptive devices and women using oral
contraceptives.
13.SERUM CALCIUM, PHOSPHURUS:
-When lesions of jaw bone are discovered on dental

radiographic examination and systemic bone disease such as
Paget’s disease, fibrous dysplasia, primary and secondary

hyperparathyroidism, osteoporosis, multiple myeloma,

osteogenic sarcoma or metastatic malignancy are suspected,
Serum calcium, Phosphate and Serum alkaline phosphatase
levels as initial screening procedures done.
-Normal value:
Serum calcium: 8.8 to 10.5 mg/dl of blood
Serum phosphorus: 2 to 5 mg/dl of blood
14. SERUM ALKALINE PHOSPHATASE:
-Alkaline phosphatase occurs in many tissues of the body, but

notably in osteoblasts.
-Normal value: 1 to 4 units/dl of blood
- Increase in serum alkaline phosphatase is seen primarily as a

result of obstructive liver disease and a variety of miscellaneous
conditions such as malignancy or abscess of the liver, amyloid
disease, leukaemia and sarcoidosis.
15. SERUM URIC ACID:
-Uric acid is a metabolic end product of nucleoprotein

metabolism derived from the purine molecule.
-Normal value: male – 4 – 8.5 mg/dl
Female - 2.8 – 7.5 mg/dl
-Increase may result from increased dietary intake, but

usually seen with gout or acute phase of disease such as
leukaemia, lymphomas, anemia or lobar pneumonia.
-Measurement of uric acid is important in the evaluation of

intrinsic disease of temporomandibular joint, particularly when

nodules consistent with gouty tophi are noted about the face
or ears.
16. SERUM ALBUMIN AND GLOBULIN:
- Serum albumin and a number of proteins involved in

coagulation are synthesized in the liver, globulin are produced
by cells of the plasma cell series.
- Because albumin and globulin constitute the major proteins of

serum, liver dysfunction may be detected by a change in the
relative concentrations of serum albumin and globulin.
17. SERUM BILIRUBIN:
- Serum Bilirubin derived from red blood cells broken down at

the end of their normal 120 days life span.
- Normal range: 0.1 – 0.8 mg/dl
- In jaundice total serum bilirubin rises above 2 to 3 mg/dl
- It may indicate the presence of hepatitis which can constitute

an infectious hazard for a dentist and his patients.
COMPOTION OF BLOOD
BLOOD CHEMISRY
FUNCTION OF BLOOD
VARIOUS BLOOD DISORDERS
SPECIAL
-ASO
ELISA
MONOSPOT PAUL
VDRL,FTE-ABS,TPI,TPHA
NAPIER’S

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BLOOD INVESTIGATIONS

DR. MAULIKA PATEL

-> Capillary Blood is collected from

- Tip of the Finger

- Hill or toe in infants

- Equipments: Spirit or 70% Alcohol, Cotton, Needle or Mechanical

DR. MAULIKA PATEL Page 2

- Puncture the site with quick stabbing motion by sterile needle or

2. Venous Blood Specimen

Venous blood is collected when investigations required large amount

-> Venous Blood is collected from

- Anterior Cubital vein

- External jugular or femoral vein in children

- Anterior Fontanelle in infants.

DR. MAULIKA PATEL Page 3

Equipments: Spirit or 70% alcohol, Cotton, Sterile Syringe & Needle,

a. Plain Test tube is used when investigations to be

DR. MAULIKA PATEL Page 4

Total RBC (Erythrocyte) Count

- The Mature Human erythrocyte is a circular, biconcave,

- 6.7 to 7.7 mm in diameter.

- Normal value: male – 4.5 – 5.9 million\mm3

female- 4.5-5.1 million\mm3

- RBCs is reduces in anaemia.

- RBCs increases in polycythaemia.

-MCV (mean corpuscular volume)

Average Red cell volume referred to as MCV is expressed in

Norma value: 80-96 cu microns

-MCH (mean corpuscular haemoglobin)

The haemoglobin contain of erythrocyte is referred to as MCH,

MCH = Haemoglobin Concentration (g\dl) X 10

Normal value: 27-31 pg

-MCHC (mean corpuscular haemoglobin concentration)

The concentration of haemoglobin is referred as MCHC:

MCHC = Haemoglobin Concentration X 100

DR. MAULIKA PATEL Page 6

Normal value: 32-36 g\dl

Types of anaemia MCV MCH MCHC

Microcytic hypochromic Decreased Decreased Decreased

Macrocytic normochromic Increased Increased Normal

Normocytic normochromis Normal Normal Normal

1. Hematocrit (Packed cell volume)

- Two heparinised micro-hematocrit capillary tubes.

- A box of matches and micro-hematocrit centrifuge equipped with

- Blood is obtained by a finger prick or venipuncture and is allowed to

- The volume of blood should be free from air bubbles.

DR. MAULIKA PATEL Page 7

- The centrifuge is started and it accelerates to 12,500 rpm and stops

- Increased in the value indicates primary and secondary

-Haemoglobin is the red pigment of blood.

-It is a chromaprotein consisting of two parts. Globin (96%) is a

-In human probably two varieties of Haemoglobin:

a. Foetal Haemoglobin (HbF) &

b. Adult Haemoglobin (HbA)

-Normal Value: male- 14 - 17.4 gm\dl

Female- 12.3 to 15.3 gm\dl

DR. MAULIKA PATEL Page 8

-Different methods used are Drabkin’s technique in which

-The haemoglobin concentration is usually measured on anti-